Project Summary/Abstract A major non-infectious complication from blood transfusions is the development of transfusion-related acute lung injury (TRALI). Although we have made progress in decreasing the incidence of TRALI through donor management, it remains the number one cause of transfusion-related mortality in the United States and a major cause of in-hospital morbidity. In this application, we will study the mechanisms by which activated platelets promote lung damage in TRALI, and we will also study how the lung in turn is the major organ involved in platelet biogenesis. In Aim 1, we will determine the mechanisms of lung injury development using a two-event mouse model of antibody-mediated TRALI. The role of mitochondrial DNA as a novel priming agent will be tested in the mouse model and in plasma samples from human subjects who developed TRALI. We will then determine the critical site(s) of cognate antigen expression using a new mouse tool to conditionally delete MHC Class I in cells of interest. Next, the downstream immune response to TRALI will be investigated by focusing on platelet-induced neutrophil extracellular trap (NET) formation and novel regulators of this pathway. By mapping critical determinants of immune priming, cell-specific cognate antigen expression, and NET-mediated lung barrier disruption, we will improve our understanding of TRALI mechanisms. In Aim 2, we will turn our attention to the contribution of the lung to platelet biogenesis during homeostasis and lung injury (TRALI). Platelets are critical to the innate immune response in lung injury, but we also hypothesize that the lung is a major site of platelet biogenesis. Using fluorescent-reporter mice, intravital microscopy, and flow cytometry, we will determine the anatomic location and activity of megakaryocytes in the lung. Platelet production from intravascular megakaryocytes in the lung will be measured during homeostasis and injury. The function of extravascular megakaryocytes in the lung will be tested using lung transplantation and intravital microscopy techniques. These experiments will establish the lung as a principle sit of platelet production. In Aim 3, we will determine the role of the lung as a niche for megakaryocytes and hematopoietic precursors. Using advanced flow-based sorting, we will test for the presence of hematopoietic precursors in the lung compared to the bone marrow. The function of these hematopoietic precursors will then be tested using reconstitution strategies involving lung transplantation and adoptive transfer of flow-sorted cells from the lung. Finally, the homing or niche-promoting factors influencing lung hematopoietic precursors will be tested. The results from this proposal will elucidate mechanisms responsible for platelet-mediated lung injury and will identify novel treatment or preventive pathways. These studies will also establish the functional role of the normal and injured lung in platelet biogenesis and hematopoietic cell maintenance, which has far-reaching public health implications.